Mitochondrial DNA Instability and Peri-Implantation Lethality ...

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/01/$04.0010 DOI: 10.1128/MCB.21.2.644–654.2001

Jan. 2001, p. 644–654 Vol. 21, No. 2

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Mitochondrial DNA Instability and Peri-Implantation LethalityAssociated with Targeted Disruption of Nuclear

Respiratory Factor 1 in MiceLEI HUO AND RICHARD C. SCARPULLA*

Department of Cell and Molecular Biology, Northwestern Medical School,Chicago, Illinois 60611

Received 6 October 2000/Accepted 16 October 2000

In vitro studies have implicated nuclear respiratory factor 1 (NRF-1) in the transcriptional expression ofnuclear genes required for mitochondrial respiratory function, as well as for other fundamental cellularactivities. We investigated here the in vivo function of NRF-1 in mammals by disrupting the gene in mice. Aportion of the NRF-1 gene that encodes the nuclear localization signal and the DNA-binding and dimerizationdomains was replaced through homologous recombination by a b-galactosidase–neomycin cassette. In themutant allele, b-galactosidase expression is under the control of the NRF-1 promoter. Embryos homozygousfor NRF-1 disruption die between embryonic days 3.5 and 6.5. b-Galactosidase staining was observed ingrowing oocytes and in 2.5- and 3.5-day-old embryos, demonstrating that the NRF-1 gene is expressed duringoogenesis and during early stages of embryogenesis. Moreover, the embryonic expression of NRF-1 did notresult from maternal carryover. While most isolated wild-type and NRF-11/2 blastocysts can develop furtherin vitro, the NRF-12/2 blastocysts lack this ability despite their normal morphology. Interestingly, a fractionof the blastocysts from heterozygous matings had reduced staining intensity with rhodamine 123 and NRF-12/2 blastocysts had markedly reduced levels of mitochondrial DNA (mtDNA). The depletion of mtDNA didnot coincide with nuclear DNA fragmentation, indicating that mtDNA loss was not associated with increasedapoptosis. These results are consistent with a specific requirement for NRF-1 in the maintenance of mtDNAand respiratory chain function during early embryogenesis.

The electron transport and oxidative phosphorylation sys-tem in mammalian mitochondria requires contributions fromboth the nuclear and the mitochondrial genetic systems. Themitochondrial DNA encodes 13 respiratory subunits, as well asthe 22 tRNAs and 2 rRNAs required for their mitochondrialtranslation. However, most respiratory proteins and all of thegene products required for mitochondrial DNA (mtDNA) rep-lication and transcription are nucleus encoded (reviewed inreferences 39 and 42). Nuclear respiratory factor 1 (NRF-1)was identified as a nuclear transcription factor that transacti-vates the promoters of a number of mitochondrion-relatedgenes in vitro (7, 10, 11, 48). These include respiratory sub-units, the rate-limiting heme biosynthetic enzyme, and factorsinvolved in the replication and transcription of mtDNA (re-viewed in reference 39). Among the most intriguing is TFAM,a nucleus-encoded transcription factor that acts on bidirec-tional promoters within the mitochondrial D-loop regula-tory region (12). TFAM was recently shown to be essentialfor mitochondrial biogenesis during embryonic development(29) and for normal function of the heart (50). Moreover,NRF-1 is involved in the transcriptional control of mito-chondrial biogenesis during adaptive thermogenesis throughits interaction with the cold-inducible coactivator PGC-1(53).

In addition to its proposed role in respiratory chain expres-

sion, NRF-1 has also been implicated in other cellular func-tions. Most recently, genes encoding two rate-limiting enzymesin purine nucleotide biosynthesis (8), a receptor involved inchemokine signal transduction (52), a subunit of a neural re-ceptor (33), and the human poliovirus receptor CD155 (44)were all shown to have functional NRF-1 binding sites in theirpromoters. These observations are consistent with a broaderrole for NRF-1 in the integration of diverse cellular functions.

Although many transcription factors are members of genefamilies, NRF-1 is a single-copy gene in vertebrates, and nofamily members have yet been detected. However, two inver-tebrate factors, P3A2 and EWG (erect wing gene product),have high sequence similarity to the NRF-1 protein that isrestricted to the DNA-binding domain (9, 48). Although P3A2acts as a negative regulator of a cytoskeletal actin gene (54),target genes for EWG have yet to be identified (9). In contrast,the NRF-1 homologue in zebrafish, not really finished (nrf), has91% identity to the human protein and was recently disruptedin vivo by insertional mutagenesis (3). Each of these genes hasbeen implicated in embryonic or larval development and botherect wing and nrf have been associated with the central ner-vous system.

We describe here the targeted disruption of the NRF-1 genein mice. The results establish that NRF-1 is essential for earlyembryogenesis in mammals, and its loss of function results ina peri-implantation lethal phenotype. In addition, NRF-12/2 blastocysts show a dramatic decrease in the amount ofmtDNA. The results are in keeping with the proposed role forNRF-1 in the maintenance of the respiratory apparatus.

* Corresponding author. Mailing address: Department of Cell andMolecular Biology, Northwestern University Medical School, 303 EastChicago Ave., Chicago, IL 60611. Phone: (312) 503-2946. Fax: (312)503-0798. E-mail: [email protected].

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MATERIALS AND METHODS

Construction of the targeting vector. The 59 homologous region was derivedfrom two genomic fragments. A 5-kb EcoRI-Acc65I genomic fragment containsan intron between exons 1 and 2. An adjacent 1-kb fragment contains the first 33nucleotides (nt) of CE2 terminated at its 39 end with a site-directed Acc65I site.Ligation of these fragments generated the 6-kb 59 homology region of thetargeting vector. The 39 homologous region encompasses a 3.2-kb XhoI-XbaIgenomic fragment beginning with the 39-terminal 108 nt of CE4 and extendinginto the intron between CE4 and CE5. The b-galactosidase–neomycin cassetteseparates truncated coding exons 2 and 4, and the herpes simplex virus thymidinekinase selectable marker lies outside of the homologous regions as depicted inFig. 1A.

Details of the vector construction are as follows. mouse P1 clone 11713 iso-lated from a 129/OLA ES cell library (23) was probed with DNA fragmentscontaining different human NRF-1 coding exons (CE) in Southern blots. pSK-mNRF1Sa10.5 was generated by cloning a 10.5-kb SacI fragment hybridizing toCE1, -2, and -3 into pBluescript SK(1) (Stratagene). pSK-mNRF1Xh10.5 wasgenerated by cloning a 10.5-kb XhoI fragment hybridizing to CE4 into pBlue-script SK(1). From pSK-mNRF1Sa10.5, a 5-kb EcoRI-Acc65I intron fragment

between CE1 and CE2 was cloned into pBluescript SK(1), resulting in pSK-mNRF1EA5. A 2-kb Acc65I-EcoRI fragment containing CE2 from pSK-mNRF1Sa10.5 was cloned into M13mp18, and an Acc65I site was created 31 bpdownstream of the 59 end of CE2 by oligonucleotide-mediated site-directedmutagenesis (27), using primer mN1cDNA366MS (59-CTGCTGTGGTACCAGGGAAGAAAC-39). The 1-kb Acc65I fragment containing 33 bp of the CE2sequence was digested from the resulting construct and cloned into pBluescriptSK(1) to generate pSK-mNRF1Ac1M. pSK-mNRF1Xh10.5 was digested withXbaI, and the 6.2-kb fragment hybridizing to CE4 was self-ligated to generatepSK-mNRF1XX3.2. This construct contained 3 kb of pBluscript SK(1) vectorsequence and a 3.2-kb insert between the XhoI and XbaI sites of the vector. The5-kb EcoRI-Acc65I fragment from pSK-mNRF1EA5 and the 3.5-kb Acc65I-XbaIfragment from pSV-b-Galactosidase Control Vector (Promega) were cloned in athree-way ligation into EcoRI-XbaI-digested pPNT (46), generating pPNT-59in-tron-LacZ. Then pPNT-59homol-LacZ was generated by cloning the 1-kb Acc65Ifragment from pSK-mNRF1Ac1M into the Acc65I site of pPNT-59intron-LacZ.pPNT-59homol-LacZ was sequenced with primer mN1INTRON3S (59-CAGTGTGTTGCTGTGTCTCTCC-39) to ensure that the remaining CE2 sequence andthe b-galactosidase sequence were in frame. The 3.2-kb insert in pSK-

FIG. 1. Targeted disruption of the mouse NRF-1 gene. (A) Schematic representation of the wild-type and the mutant mouse NRF-1 allelescovering CE1 to CE5 (filled boxes). The bold solid lines represent introns, and the thin solid lines indicate the plasmid backbone in the targetingvector. A promoterless b-galactosidase gene cassette (b-gal, hatched box) was inserted downstream of the first 33 bp of CE2 in the targeting vector.The neomycin cassette (neo, vertical hatched box) and the herpes simplex virus thymidine kinase cassette (hsv-tk, open box) each contain a mousephosphoglycerate kinase promoter, and their transcriptional orientations are from right to left. The 59 and 39 homologous regions are 6 and 3.2kb, respectively. Upon homologous recombination, the 5.3-kb b-gal-neo sequence replaces approximately 7 kb of endogenous NRF-1 genesequence. Restriction enzyme cleavage sites shown above and below: B, BamHI; S, SacI; X, XbaI. The positions of probes used for genotyping bySouthern blot analysis are indicated above the wild-type allele. Exons and the intron between CE4 and CE5 are not drawn to scale. (B) Southernblot analysis used to screen ES clones and genotype the progeny from heterozygous matings. The restriction enzymes and probes are shown aboveand below, respectively. The sizes of the hybridizing fragments in the wild-type (wt) and mutant (mut) alleles are shown on the right. (C) PCRprimers for genotyping. The positions of the primers are indicated by solid bars. Primers 1 to 4 were designed to detect the wild-type allele, andprimers 5 to 8 were designed to detect the mutant allele. In CE2, the sequence to the right of the dotted line is deleted in the mutant allele. Primers1, 2, 5, and 6 are in the sense orientation, and primers 3, 4, 7, and 8 are in the antisense orientation. (D) PCR genotyping of the progeny fromheterozygous matings. As described in Materials and Methods, newborn mice and 6.5- to 8.5-day-old embryos were genotyped with primers 2, 4,5, and 7 shown in panel C, while preimplantation embryos were genotyped with primers 1 to 8 by a nested-PCR method. The sizes of the PCRproducts are indicated on the right of each panel.

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mNRF1XX3.2 was cut out with XhoI and NotI and cloned into pPNT-59homol-LacZ between its XhoI and NotI sites. This resulted in the 20-kb targeting vector,named pmNRF1KO.

Generation of targeted ES cells and mutant mice. pmNRF1KO was linearizedwith NotI prior to electroporation. Embryonic stem (ES) cell culture, electropo-ration, blastocyst injection, and generation of germ line-transmitting foundermice were carried out by the Targeted Mutagenesis Facility in the Children’sMemorial Institute for Education and Research at Northwestern University.

Screening of ES clones and genotyping of newborn mice and 6.5- to 8.5-day-oldembryos. The following oligonucleotides were used in screening and genotyping,as depicted in Fig. 1C: mN1cDNA385S (primer 1), 59-GAAACGGAAACGGCCTCATGTG-39; mN1cDNA419S (primer 2), 59-CCATCTATCCGAAAGAGACAGCAGAC-39; mN1INTRON4AS (primer 3), 59-CCTCAAGACACTGGCATGG AG-39; mN1INTRON4AS2 (primer 4), 59-AGGTTTAGACTTGGAATCACTCCCGT-39; pPNT778S-neo (primer 5), 59-TGAATGAACTGCAGGACGAGG-39; pPNT841S-neo (primer 6), 59-CAGCTGTGCTCGACGTTGTCA-39;pPNT1237AS-neo (primer 7), 59-CCACAGTCGATGAATCCAGAA-39; andpPNT1279AS-neo (primer 8), 59-GCCAACGCTATGTCCTGATAG-39.

Southern blot analysis was performed to identify ES clones that had under-gone homologous recombination. SacI-digested and XbaI-digested ES cell DNAwas probed with probe 1, a 200-bp DraIII-PstI genomic fragment containingmouse NRF-1 coding exon 1 sequence (Fig. 1A). BamHI-digested ES cell DNAwas probed with probe 2, a 570-bp SacI-HindIII mouse genomic fragment hy-bridizing to human NRF-1 cDNA nt 761 to 1020 (48). The sizes of the hybrid-izing fragments from the wild-type and mutant alleles are indicated in Fig. 1B.Tails from newborn mice and 6.5- to 8.5-day-old embryos were genotyped byPCR with PTC-100 programmable thermal controller (M. J. Research, Inc.). A183-bp NRF-1 sequence absent in the mutant allele was amplified to identify thewild-type allele, and a 460-bp neomycin fragment was amplified from the mutantallele as shown in Fig. 1D. Tails from about 100 newborn mice from threegenerations were also genotyped by Southern blot as described above to verifythe accuracy of the PCR method. PCR was performed using AmpliTaq poly-merase (Perkin-Elmer) with 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphos-phates (dNTPs) 0.3 mM concentrations of primers pPNT778S-neo andpPNT1237AS-neo, and 0.1 mM concentrations of primers mN1cDNA419S andmN1INTRON4AS2 in a total volume of 25 ml. The cycling conditions were 94°Cfor 30 s, 55°C for 30 s, and 72°C for 30 s for 30 cycles.

Genotyping of preimplantation embryos. We isolated 2.5- and 3.5-day-oldembryos by flushing mouse uteri, and each was collected in 10 to 15 ml ofphosphate-buffered saline (pH 7.2). DNA was isolated by incubation at 95°C for10 min, with 20-mg proteinase K treatment at 55°C for 3 h, followed by inacti-vation of the proteinase at 95°C for 10 min. Half of the DNA from each embryowas used for genotyping by a nested PCR method using Platinum Taq DNApolymerase (GIBCO-BRL). The first PCR was carried out with 1.5 mM MgCl2,0.2 mM dNTPs, 0.3 mM concentrations of primers pPNT778S-neo andpPNT1279AS-neo, and 0.1 mM concentrations of primers mN1cDNA385S andmN1INTRON4AS2 in a total volume of 25 ml. The cycling conditions were 94°Cfor 30 s, 55°C for 30 s, and 72°C for 30 s for 25 cycles. The second PCR wascarried out with 1 ml of product mixture from the first PCR, 0.3 mM concentra-tions of primers pPNT841S-neo and pPNT1237AS-neo, and 0.15 mM concen-trations of primers mN1cDNA419S and mN1INTRON4AS. The cycling condi-tions were the same as for the first PCR except that the cycle number was 20.Products of the second PCR included a 136-bp fragment from the wild-type alleleand a 398-bp fragment from the mutant allele as depicted in Fig. 1D.

RNase protection assays. To generate the mouse NRF-1 riboprobe, primersmN1cDNA671S (59-CTGCCGCCTCTCACCATCGAT-39) and mN1cDNA1016AS (59-GATGAGCTATACTGTGTGTGGTG-39) were used to PCR-am-plify a 346-bp NRF-1 cDNA fragment located 39 of the deleted region. The PCRproduct was cloned into pGEM-T (Promega), and a 580-bp antisense riboprobewas synthesized with Sp6 RNA polymerase after the plasmid DNA was linearizedwith PvuII. To generate the NRF-1–b-galactosidase riboprobe, a 192-bp frag-ment was amplified with genomic DNA isolated from NRF-1 heterozygous mice,using primers mN1INTRON3S and pSVbgal710AS (59-CGGGATCGATCTCGCCATACA-39). The product, containing 69 bp of NRF-1 intron sequence, thefirst 33 bp of NRF-1 CE2 sequence and 90 bp of sequence from pSV-b-Galac-tosidase Control Vector, was cloned into pGEM-T. The plasmid DNA waslinerized with MluI, and a 292-bp antisense riboprobe for the NRF-1–b-galac-tosidase fusion gene in the mutant allele was generated with T7 RNA polymer-ase. Hybridization was performed as described previously (23), with 10 mg oftotal RNA from mouse tissues or yeast tRNA as a negative control.

b-Galactosidase staining of embryos. We stained 2.5- and 3.5-day-old embryosfor b-galactosidase activity as described previously (47). Briefly, freshly isolatedembryos were fixed for 10 min in 1% (vol/vol) paraformaldeyde, 0.2% (vol/vol)

glutaraldehyde, and 1% (vol/vol) calf serum in phosphate-buffered saline (pH7.2). They were then rinsed in phosphate-buffered saline (pH 7.2) and trans-ferred to a mixture containing 0.02% NP-40, 0.01% sodium deoxycholate, 5 mMK4Fe(CN)6 z 3H2O, 5 mM K3Fe(CN)6, 2 mM MgCl2, and 1 mg of 4-chloro-5-bromo-3-indolyl-b-galactoside (X-Gal) per ml in phosphate-buffered saline (pH7.3). Positively stained embryos were scored after 20 h of incubation at 37°C.

b-Galactosidase staining of the ovary. Ovaries were isolated freshly fromsexually mature mouse females. They were fixed in phosphate-buffered saline(pH 7.2) containing 2% paraformaldehyde, 0.02% glutaraldehyde, and 2 mMMgCl2 for 90 min at 4°C and then equilibrated in 10% sucrose–2 mM MgCl2 inphosphate-buffered saline (pH 7.2) for 2 h at 4°C and in 20% sucrose–2 mMMgCl2 in phosphate-buffered saline (pH 7.2) for 2 h at 4°C. Samples were thenembedded in OCT and frozen at 280°C. Cryostat sections (8 mm) were made,and the slides were stained for b-galactosidase activity as described previously(21). Briefly, the slides were first treated with 0.02% NP-40, 0.01% sodiumdeoxycholate, and 2 mM MgCl2 in phosphate-buffered saline (pH 7.3) for 5 minat 25°C. Samples were then fixed in phosphate-buffered saline (pH 7.2) contain-ing 2% paraformaldehyde, 0.02% glutaraldehyde, and 2 mM MgCl2 for 5 min at25°C and stained in 0.02% NP-40, 0.01% sodium deoxycholate, 5 mM K4Fe(CN)6 z 3H2O, 5 mM K3Fe(CN)6, 2 mM MgCl2, and 1 mg of X-Gal per ml inphosphate-buffered saline (pH 7.3) for 20 h at 37°C in 5% CO2 in a humidifiedincubator. The slides were rinsed in phosphate-buffered saline (pH 7.2) and H2Oand stained in 0.25% eosin–85% ethanol for 1 min at room temperature forbetter visualization of the staining.

Embryo culture. To culture blastocysts in vitro, Dulbecco modified Eaglemedium (GIBCO-BRL 11965-092) was supplemented with 15% (vol/vol) fetalbovine serum (HyClone), 100 U of penicillin per ml, 100 mg of streptomycin perml, 2 mM L-glutamine, 0.1 mM minimal essential medium with nonessentialamino acids, 8 mg of adenosine per ml, 8.5 mg of guanosine per ml, 7.3 mg ofcytidine per ml, 7.3 mg of uridine per ml, 2.4 mg of thymidine per ml, 0.1 mMb-mercaptoethanol, and 200 U of murine leukemia inhibitory factor (GIBCOBRL) per ml (1). Blastocysts were cultured individually in microdrops undermineral oil at 37°C in 5% CO2 in a humidified incubator. They were examinedafter 40 and 100 h in culture and were genotyped by the nested-PCR methoddescribed above.

Rhodamine 123 staining of blastocysts. Freshly isolated blastocysts were in-cubated in 10 mg of rhodamine 123 per ml and 1% calf serum in phosphate-buffered saline (pH 7.2) for 30 min at 37°C (24). They were then washed inphosphate-buffered saline (pH 7.2) and viewed under a fluorescent microscopeat 485 nm.

mtDNA copy number analysis in blastocysts. For mtDNA copy number anal-ysis, half of the DNA from each genotyped blastocyst was used to amplify a648-bp mouse mt DNA fragment corresponding to nt 267 to 914 of the cyto-chrome oxidase subunit 1 gene and a 316-bp 5S ribosomal DNA (rDNA) frag-ment in a PCR with Platinum Taq DNA polymerase. The 5S rDNA fragmentcorresponded to nt 699 to 1014 in the GenBank sequence database underaccession number D17317. The PCR was carried out with 1.5 mM MgCl2, 0.2mM dNTPs, 0.3 mM concentrations of primers m5SrDNA699S (59-ACCGTCTAGCCGTCCTCCTT-39) and m5SrDNA1014AS (59- CCCACTGAGGATGGATACATG-39), and 0.15 mM concentrations of primers mCO1-267S (59- CCCAGATATAGCATTCCCACGA-39) and mCO1-914AS (59-AGCAAGCTCGTGTGTCTACATC- 39). The cycling conditions were 94°C for 30 s, 55°C for 30 s, and72°C for 30 s for 25 cycles. To generate standards for comparison, PCRs werealso performed under the same conditions, in each experiment, with a series oftwofold dilutions of heart DNA isolated from an adult wild-type mouse astemplates. The products from the standards and tested blastocysts were resolvedon a 1.2% agarose gel. The mtDNA fragment was visualized by ethidium bro-mide staining. The 5S rDNA fragment was detected by autoradiography follow-ing Southern blotting.

mtDNA copy number analysis in unfertilized eggs. Superovulation was in-duced as previously described (21). Female mice that were 3 to 5 weeks old andweighed between 12 and 16 g were injected intraperitoneally with 5 IU ofpregnant mare’s serum (Sigma G4877), followed by an intraperitoneal injectionof 5 IU of human chorionic gonadotropin (Sigma C1063) 45 h later. They weresacrificed the next day, and unfertilized eggs were obtained from the oviducts.The cumulus cells surrounding the eggs were removed by incubation in 300 mg ofhyaluronidase per ml in phosphate-buffered saline (pH 7.2) for 5 to 10 min. Eggsfrom the same female were pooled in phosphate-buffered saline (pH 7.2) at aconcentration of 1 egg/ml. DNA was isolated as described above for blastocysts.To analyze the amount of mtDNA in unfertilized eggs from wild-type andheterozygous females, The total DNA amounts isolated from 2.5, 5, or 10 wild-type eggs were used as PCR templates to generate standards, and the total DNAamounts from 5 eggs of heterozygous females were used as templates for com-

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parison. The same mtDNA fragment and 5S rDNA fragment described abovefor the blastocysts were amplified in a PCR with Platinum Taq DNA poly-merase. The PCR was carried out with 1.5 mM MgCl2, 0.2 mM dNTPs, 0.3 mMconcentrations of primers m5SrDNA699S and m5SrDNA1014AS, and 0.1 mMconcentrations of primers mCO1-267S and mCO1-914AS. The cycling conditionswere 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 22 cycles. The productswere resolved on a 1.2% agarose gel, the mtDNA fragment was visualized byethidium bromide staining, and the 5S rDNA fragment was detected by autora-diography following Southern blotting.

TUNEL staining of 3.5-day-old embryos. We stained 3.5-day-old embryos forDNA strand breaks by TUNEL (terminal deoxynucleotidyl-transferase-mediateddUTP-biotin nick end labeling) method using the In Situ Cell Death DetectionKit, Fluorescein (Boehringer Mannheim). Briefly, freshly isolated embryos werefixed in 4% (vol/vol) paraformaldehyde and 1% (vol/vol) calf serum in phos-phate-buffered saline (pH 7.2) for 5 min and then permeabilized in 0.1% TritonX-100, 0.1% sodium citrate, and 1% (vol/vol) calf serum in phosphate-bufferedsaline (pH 7.2) for 5 min on ice. They were then rinsed in phosphate-bufferedsaline (pH 7.2) and incubated in the TUNEL reaction mixture for 1 h at 37°C.Positive controls were treated the same except that before incubation in thereaction mixture, they were incubated in phosphate-buffered saline (pH 7.5)containing 50 U of RQ1 DNase (Promega) per ml, 10 mM Tris-HCl, 1 mMMgCl2, and 1 mg of bovine serum albumin per ml for 30 min at 37°C. Stainedembryos were viewed under a fluorescent microscope at 485 nm and then col-lected for genotyping as described above.

RESULTS

Targeted disruption of the mouse NRF-1 gene. To disruptthe mouse NRF-1 gene, a targeting vector was constructed inwhich the NRF-1 sequence encoding amino acids 86 to 166 andencompassing coding exons 2 through 4 was replaced by ab-galactosidase–neomycin cassette (Fig. 1A). This region con-tains the nuclear localization signals and the DNA-binding anddimerization domains, and its deletion upon homologous re-combination should completely eliminate NRF-1 function (15,16, 48). In the mutant allele, the b-galactosidase coding se-quence is fused in frame with the first 85 amino acids ofNRF-1, thus placing b-galactosidase expression under the con-trol of the NRF-1 promoter and 59-untranslated region. Thetargeting vector was electroporated into 129/SvJ-derived (ES)cells, and G418 and ganciclovir double-resistant cells werescreened. Homologous integration into the NRF-1 locus wasconfirmed in approximately 20% of the selected ES clones(Fig. 1B) by Southern blot hybridization. Five positive ESclones were microinjected into C57BL/6 blastocysts, and threechimeras from one of the clones showed germline transmis-sion. Most of the heterozygous NRF-1 mice were viable andfertile. They were interbred, and their offspring were geno-typed by Southern blotting and PCR analysis (Fig. 1B to D).Among the initial 412 newborns generated by heterozygousmatings, none were homozygous mutants, and the ratio ofheterozygous to wild-type offspring was 1.78 (Table 1). Thiswas indicative of an embryonic lethal phenotype associatedwith the NRF-12/2 genotypes.

The stage of embryonic death associated with NRF-1 loss of

function was investigated by determining the genotypes of em-bryos between 6.5 and 8.5 days postcoitus (dpc). Surprisingly,no homozygous NRF-1 embryos were identified at this stage(Table 1). Therefore, a nested-PCR strategy was developed togenotype preimplantation embryos (Fig. 1C and D). Among 68blastocysts isolated at 3.5 dpc, 15 were wild-type, 35 wereheterozygous, and 18 were homozygous mutant (Table 1). Thisresult is consistent with the expected ratio for Mendelian in-heritance. Thus, homozygous null mutations in NRF-1 result inlethality between embryonic days 3.5 and 6.5.

NRF-1 expression in preimplantation embryos. The deathof NRF-12/2 embryos around the time of implantation in-dicated that NRF-1 function was required for developmentbeyond this stage. It was demonstrated recently that NRF-1expression can be detected in 7.5-dpc mouse embryos by North-ern analysis (40). However, it is unknown whether NRF-1 isexpressed earlier. The targeting vector was designed with theexpectation that, by fusing b-galactosidase in frame withNRF-1 coding exon 2 (Fig. 1A), b-galactosidase activity couldbe measured as a good approximation of NRF-1 expression.To confirm this, RNase protection assays were performed todetect the expression of NRF-1–b-galactosidase fusion tran-script relative to that of the endogenous NRF-1 transcript intissues obtained from NRF-12/2 mice (Fig. 2A). Total RNA(10 mg) isolated from various tissues was hybridized simulta-neously to a specific NRF-1 riboprobe and to an NRF-1–b-galactosidase fusion gene riboprobe. The results demonstratedthat the relative expression of the fusion gene in various tissuesclosely paralleled that observed for the NRF-1 transcript itself,with the highest expression in the testis and the lowest expres-sion in the heart and liver (lanes 1 to 5). The heterogeneity ofprotected fusion gene transcripts (approximately 120 nt) didnot result from degradation of the NRF-1 transcript becausethe fusion gene riboprobe alone yielded the same heteroge-neous protected bands upon hybridization to kidney RNAfrom NRF-11/2 animals (lane 6). The specificity of the fusiongene riboprobe was tested by hybridizing total kidney RNAfrom a wild-type mouse to both riboprobes. In this case, onlythe protected NRF-1 transcript from the wild-type allele wasobserved (lane 7). The results support the conclusion that theexpression of the b-galactosidase gene is directed from theNRF-1 promoter and therefore reflects NRF-1 expression.

To determine whether NRF-1 is expressed in preimplanta-tion embryos, 2.5- and 3.5-dpc embryos were isolated, andb-galactosidase staining was performed. The results showedthat 17 of 23 blastocysts (74%) and 15 of 19 eight-cell morulae(79%) obtained from heterozygous matings stained positive forb-galactosidase activity (Fig. 2Bc and f; Table 2), whereas allembryos from wild-type crosses were negative (Fig. 2Ba and d).In addition, representative stained embryos were genotyped.All positively stained embryos were either NRF-12/2 or NRF-11/2, and all negatively stained ones were wild type (not shown).This is consistent with the expected expression of b-galactosi-dase activity from the endogenous promoter.

Active gene expression has been demonstrated during oo-cyte growth at the stage of the first meiotic prophase, andtranscripts and proteins expressed during this time could becarried over to early embryos (reviewed in references 41 and51). If the b-galactosidase activity observed in NRF-1 embryosresults from the inheritance of maternal gene products, one

TABLE 1. Genotypes of progeny from heterozygous matings

Stage(dpc)

No. of progeny (%) with genotype:Total no.

1/1 1/2 2/2

Newborna 148 (36) 264 (64) 0 4126.5–8.5 27 (38) 45 (62) 0 723.5 15 (22) 35 (51) 18 (26) 68

a DNA was isolated from 1-day-old to 3-week-old animals.



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would expect to see positive staining in all embryos from het-erozygous crosses because all wild-type haploid eggs from het-erozygous females should also obtain the gene products ex-pressed before the first meiotic division. The observation thatwild-type 2.5- and 3.5-dpc embryos from heterozygous matingsstained negative for b-galactosidase activity thus argues againstmaternal inheritance of b-galactosidase. To confirm this, wild-type females were crossed to heterozygous males, and embryoswere isolated for b-galactosidase staining. Since wild-type fe-males do not express any bacterial b-galactosidase, any de-tected activity would thus be expressed from the mutant alleleobtained from the male. The results show that 5 of 12 blasto-cysts (42%) and 6 of 15 eight-cell morulae (40%) from suchmatings stained positive for b-galactosidase activity (Fig. 2Bband e; Table 2). Therefore, NRF-1 is expressed in preimplan-tation embryos at no later than the eight-cell morular stage.

NRF-1 is required for growth of blastocysts in vitro. Themorphology of NRF-12/2 blastocysts was indistinguishablefrom that of NRF-11/2 or wild-type blastocysts. Given thelack of an obvious phenotype, it was of interest to determine

TABLE 2. b-Galactosidase staining of embryos

Mating type Stage (dpc) Litter(n)

No. of embryos

Stained Notstained Total

1/2 3 1/2 Blastocyst (3.5) 3 17 6 23Eight-cell morula (2.5) 2 15 4 19

1/1F 3 1/2Ma Blastocyst (3.5) 2 5 7 12Eight-cell morula (2.5) 2 6 9 15

a F, female; M, male.

FIG. 2. b-Galactosidase expression. (A) Expression of NRF-1 and the NRF-1–b-galactosidase fusion gene transcripts in adult mice. A total of10 mg of RNA isolated from the indicated tissues of an NRF-11/2 mouse (lanes 1 to 6) or from kidney of a wild-type littermate (lane 7) wasanalyzed for the expression of NRF-1 and NRF-1–b-galactosidase transcripts by RNase protection assay. Lane 6, with the NRF-1–b-galactosidaseriboprobe alone; other lanes, with both NRF-1 and NRF-1–b-galactosidase riboprobe. The sizes of the protected products are indicated on theright. (B) b-Galactosidase expression in embryos. Embryonic stages are indicated on the left, and the various crosses are shown above. F, female;M, male. Positively stained embryos are indicated by arrows. b-Galactosidase activity was readily detected in eight-cell morulae (b and c) and inboth the inner cell mass and trophectoderm cells in blastocysts (e and f).



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whether the arrest at this stage was associated with an intrinsicdefect in cellular proliferation. To this end, blastocysts fromheterozygous matings were isolated and cultured in vitro.Among 36 blastocysts obtained from five litters, 23 showedtypical morphology in culture after 5 days, with the trophoblastspreading out on the culture dish and a proliferating inner cellmass on top (Fig. 3C; Table 3). In contrast, the other 13blastocysts were unable to attach to the culture dishes andshowed no sign of growth (Fig. 3D and E). Some of them werelost after 5 days in culture, presumably from lysis of the entireembryo. Some appeared to collapse and showed a decrease insize (Fig. 3D and E). Attempts to genotype these blastocysts byPCR were unsuccessful. The 23 embryos that grew in culturewere genotyped, among which 14 were heterozygous and 9were wild-type (Table 3). These results suggested that none ofthe homozygous NRF-1 blastocysts could grow normally invitro.

Wild-type and NRF-11/2 blastocysts were further tested fortheir ability to grow in culture. A total of 30 blastocysts (fourlitters) from wild-type matings or heterozygous-to-wild-typematings were cultured under the same conditions. Amongthem, 28 grew normally. The two that did not grow were fromheterozygous-to-wild-type matings, suggesting that a smallnumber of heterozygous blastocysts may also be defective in invitro development (Table 3). Thus, despite their normal mor-phology, NRF-12/2 blastocysts displayed a generalized defectin their ability to develop further in vitro.

Homozygous disruption of NRF-1 is associated with a de-crease in mitochondrial staining and a reduction in mtDNAcontent. Given the proposed role for NRF-1 in respiratorygene expression, it was of interest to determine whether NRF-12/2 blastocysts had a mitochondrion-related phenotype. Rho-damine 123 staining requires a normal mitochondrial mem-brane potential and has been used for fluorescent staining offunctional mitochondria with low background (24). Embryosfrom heterozygous or wild-type matings were isolated at 3.5dpc and stained with rhodamine 123. The 17 blastocysts (threelitters) obtained from wild-type matings were similar in the

overall intensity of staining (Fig. 4Aa). In contrast, among 13embryos (two litters) from heterozygous matings, five blasto-cysts had a weaker and more diffuse staining pattern than thoseof their littermates, which resembled wild-type blastocysts instaining intensity (Fig. 4Ab). Although the genotypes of thestained embryos were unknown because rhodamine 123 stain-ing interfered with genotyping, the results were consistent witha reduction in the number or function of mitochondria asso-ciated with the loss of NRF-1.

The correlation between NRF-1 and mitochondrial functionwas further examined by comparing the amount of mtDNA inNRF-12/2 and wild-type blastocysts. This was accomplished byfirst genotyping individual blastocysts and then subjecting totalDNA from NRF-12/2 and wild-type blastocysts to PCR am-plifications of a 648-bp mtDNA fragment and a 316-bp 5SrDNA fragment. The latter served as an internal loading con-trol for quantifying mtDNA levels (Fig. 4B). Standards weregenerated using adult heart genomic DNA (Fig. 4B, lanes 5 to11). In comparison, NRF-12/2 blastocysts showed a reducedamount of mtDNA ranging from 30 to ,5% of wild-type con-trols (lanes 2 to 4). This is consistent with the reduction inrhodamine staining indicative of a loss of mitochondrial func-tion. Thus, NRF-1 is required for the maintenance of normallevels of mtDNA in blastocysts.

NRF-1 expression in the ovary. During mouse oocytegrowth, the paired homologous chromosomes are fully ex-

TABLE 3. Growth of blastocysts in culture

Matingtype

Litter(n)

No. ofblastocysts

with nogrowth

No. of blastocysts withnormal growth Total

Total 1/2 1/1

1/2 3 1/2 5 13 23 14 9 361/1 3 1/2 2 2 12 NDa ND 141/1 3 1/1 2 0 16 ND ND 16

a ND, the genotypes of progeny from 1/1-to-1/2 matings and wild-typematings were not determined.

FIG. 3. Growth of blastocysts in culture. Blastocysts were cultured in vitro as described in Materials and Methods. (A) Blastocyst in culturemedium immediately after isolation (day 0 in culture). (B and D) Blastocyst in culture for 40 h (day 2). (C and E) Blastocyst in culture for 100 h(day 5). Most blastocysts attached and started to grow at day 2 (B) and showed typical morphology at day 5, including the trophoblastic giant cells(TG) and the inner cell mass (ICM) (C). Putative NRF-12/2 blastocysts failed to grow (D and E). (B and C) The grown blastocyst was genotypedas NRF-11/1. All of the images are at the same magnification.



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tended and gene expression occurs. This accounts for the ma-ternally inherited mRNAs and proteins in the early embryos. Itis also well documented that mammalian mtDNA is amplifiedat least 100-fold during oocyte growth so that the final mtDNAcopy number in the mature egg is approximately 100,000 (31,36). To determine whether NRF-1 expression coincides withmaternal gene expression and mtDNA amplification in oo-cytes, ovaries from sexually mature female heterozygotes weresectioned and stained for b-galactosidase activity. As shown inFig. 5B, both the corpus luteum and the thecal cells surround-ing the follicles were most darkly stained, while some of thefollicular cells were lightly stained. Careful examinations ofserial sections revealed that oocytes at all stages of maturation

also stained positive for b-galactosidase activity (Fig. 5C to F),with medium-sized ones being the most darkly stained (panelE). The b-galactosidase activity was cytoplasmic with a smallarea away from the nucleus stained most intensely, resulting inthe appearance of a dark blue spot in the relatively lightlystained cytoplasm (Fig. 5C, D, and F). The nucleus was un-stained, presumably because a nuclear localization signal wasabsent in the fusion protein. Ovaries from wild-type controlsstained negative (Fig. 5A), indicating that the staining resultedfrom the expression of the mutant NRF-1 allele. These resultsdemonstrate that NRF-1 is expressed throughout oocyte mat-uration where gene expression and a massive mtDNA ampli-fication occur.

Measurements of the amount of mtDNA in unfertilized eggs.It is possible that the reduced mtDNA levels observed in NRF-12/2 blastocysts is caused by the effect of NRF-1 heterozygos-ity on mtDNA amplification during oocyte growth. If this is thecase, unfertilized eggs from heterozygotes should exhibit de-creased mtDNA levels. To test this possibility, unfertilized eggswere obtained from wild-type and heterozygous female mice,and the amounts of mtDNA were compared after PCR ampli-fication of the 648-bp mtDNA fragment (Fig. 4C). Again, the5S rDNA fragment was amplified as an internal loading con-trol. When DNA isolated from five eggs was used as the PCRtemplate, roughly the same amount of mtDNA product wasobtained for wild-type and heterozygous females (lanes 2, 3,and 5). DNA isolated from 10 wild-type eggs did not generateproportionately more product than that from 5 eggs, suggest-ing that the PCR may be close to saturation (lane 6). However,DNA isolated from the equivalent of 2.5 wild-type eggs gave alinear decrease in product (lane 4), indicating that the differ-ence in the amount of mtDNA between wild-type and het-erozygous eggs is ,2-fold. Thus, mtDNA amplification in oo-cytes does not appear to be dramatically affected by the loss ofone copy of the NRF-1 gene. This indicates that the massiveloss of mtDNA observed in the blastocyst occurs after fertili-zation.

Loss of mtDNA in NRF-12/2 blastocysts is not associatedwith increased apoptosis. NRF-1 may affect apoptosis throughits regulation of mitochondrial functions. One explanation forthe loss of mtDNA in NRF-12/2 blastocysts is that it is asso-ciated with apoptotic cell death that occurs after fertilization asa consequence of the absence of NRF-1. It is well establishedthat limited apoptosis does occur during early mammalianembryogenesis starting around the blastocyst stage (18), and itis possible that NRF-1 loss leads to massive cell death and theloss of the embryo. To examine this possibility, TUNEL stain-ing was performed on both wild-type and NRF-12/2 blasto-cysts to detect the chromosomal DNA breakage associatedwith apoptosis. For comparison, a positive control was gener-ated by treating wild-type blastocysts with DNase (Fig. 6A andB). The results demonstrate that while small numbers of apo-ptotic cells are detectable in both wild-type and NRF-12/2

blastocysts, there is no difference between the two in the over-all levels of apoptotic cell death (Fig. 6C to F). This suggeststhat the loss of mtDNA is not a by-product of increased apo-ptosis but rather a consequence of the disruption of NRF-1-dependent pathways of mtDNA maintenance.

FIG. 4. Mitochondrial staining and mtDNA copy number analyses.(A) Rhodamine 123 staining of blastocysts. Blastocysts were stainedwith rhodamine 123 as described in Materials and Methods. Fourblastocysts from a wild-type mating (a) are compared to three blasto-cysts from a heterozygous mating (b). Those with weak fluorescenceare indicated by the arrows. (B) mtDNA copy number in blastocysts.Half of the DNA isolated from each of the indicated embryos (lanes 2to 4) was used to amplify a mtDNA fragment and a 5S rDNA fragmentin a PCR. For standards, genomic DNA prepared from the heart of anadult wild-type mouse was diluted and subjected to the same PCRamplification (lanes 5 to 11). The amount of template was indicated infold at the top for each standard. Products were resolved on a 1.2%agarose gel, and the mtDNA product was visualized by ethidium bro-mide staining. The 5S rDNA product was detected by Southern blot-ting. (C) mtDNA copy number in unfertilized eggs. Unfertilized eggswere isolated from wild-type or heterozygous NRF-1 females for DNApreparation as described in Materials and Methods. DNA from 5 eggsfrom a heterozygous animal (lanes 2 and 3) or from the equivalent of2.5, 5, or 10 eggs from a wild-type animal (lanes 4, 5, and 6, respec-tively) was used as the template in PCR amplifications of the samemtDNA sequence as described in panel B. The numbers of eggs fromwhich the template DNA was isolated are indicated above the panels.



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DISCUSSION

NRF-1 expression and embryonic development. We demon-strate here that homozygous disruption of the mouse NRF-1gene leads to embryonic death around the time of implanta-tion. Loss of NRF-1 also coincides with the reduction ofmtDNA in the blastocyst, thus providing the first evidence thatNRF-1 is required for mitochondrial maintenance in vivo.

The targeting vector was designed so that in the mutantallele the first 85 amino acids of NRF-1 are fused in frame tob-galactosidase and polyadenylation signals are present at the39 end of the transcriptional unit. This feature, along with theopposite transcriptional orientation of the neomycin cassette,would direct the termination of the NRF-1–b-galactosidasefusion transcript at the 39 end of the b-galactosidase gene,without any downstream NRF-1 sequence expressed. The de-

letion of amino acids 1 to 77 has no affect on the ability ofNRF-1 to transactivate the cytochrome c promoter in trans-fection assays (16). b-Galactosidase is expressed from the en-dogenous NRF-1 promoter, and the fidelity of fusion geneexpression was confirmed by the similar tissue expression pat-terns of NRF-1 and the fusion transcripts. b-Galactosidaseactivity was readily detected in growing oocytes of heterozy-gous females, as well as in heterozygous and homozygouseight-cell morulae and blastocysts. According to a thoroughinvestigation of accumulation profiles of known transcripts inpreimplantation embryos, most genes, once activated at thetwo-cell stage, continue to be transcribed at least into theblastocyst stage (reviewed in reference 25). Although b-galac-tosidase staining was not performed in embryos before theeight-cell stage, it is likely that NRF-1 expression starts at an

FIG. 5. NRF-1 expression in ovary. (A) b-Galactosidase staining of an ovary from a wild-type mouse. (B) b-Galactosidase staining of an ovaryfrom a NRF-11/2 mouse. (C to F) b-Galactosidase staining of follicles from a NRF-11/2 mouse containing eggs at various stages of maturity. Thecorpus luteum (CL), the thecal cell layer surrounding the follicle (T), the follicle (Fl), the zona pellucida (ZP, unstained), the cytoplasm (Cy), andthe nucleus (Nu) are indicated (B and E). The concentrated regions of b-galactosidase accumulation within the cytoplasm are indicated by arrowsin panels C and F. Panels A and B are at the same magnification; panels C to F are at the same magnification.



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earlier time and that its continuous expression throughoutpreimplantation development accounts for the observed b-ga-lactosidase activity in the early embryo.

It is interesting to note that the loss of NRF-1 or its relativesresults in different phenotypes in both vertebrates and inver-tebrates. P3A2 is a negative regulator of transcription in seaurchins that functions in the spatial expression of a cytoskeletalactin gene during development. Its loss of function in the eggaffects the morphogenesis of the archenteron and results inembryonic lethality prior to gastrulation (4). In Drosophilamelanogaster, the erect wing locus is required for proper neu-romuscular development, and certain alleles result in late em-bryonic or early larval lethality presumably due to nervoussystem dysfunction (13, 14). Unlike the invertebrate relatives,the sequence similarity between nrf in zebra fish and the mam-malian NRF-1 is not restricted to the DNA-binding domainbut rather is distributed throughout the coding region. Duringdevelopment, expression of nrf occurs predominantly in thecentral nervous system and the optic tracts. A homozygousinsertional disruption of the nrf locus in zebra fish results in theloss of photoreceptors in the retina and ultimately death at thelarval stage (3). In each case, loss-of-function mutations led todevelopmental arrest. However, in mice, the early lethality ofthe NRF-1-null embryos suggests that, in mammals, NRF-1expression is required to proceed beyond the small number ofcell divisions leading to the blastocyst stage.

Several recently described transcription factor gene knock-outs also exhibit a peri-implantation lethal phenotype. As ex-

amples, the stage of developmental arrest has been character-ized in vitro for B-myb (45), GATA6 (26), vHNF1 (2), and Max(43) knockouts. Blastocysts from the vHNF1 homozygous nullmutants are morphologically identical to the wild-type andexhibit normal outgrowth. In contrast, blastocysts from bothB-myb and GATA6 homozygous null mutants generate tropho-blastic giant cells in culture but are severely impaired in theproliferation of the inner cell mass. Blastocysts from Max ho-mozygous null mutants are more severely impaired in that theyappear normal initially but outgrowth ceases after 2 to 3 daysin culture. NRF-12/2 blastocysts differ markedly from all ofthese cases in that morphologically normal blastocysts form invivo, but they do not attach when placed in culture and thereis no proliferative outgrowth. Interestingly, the timing of thisarrest coincides approximately with the resumption of mtDNAreplication that occurs around the time of implantation (30).

NRF-1 and mitochondrial function in early embryogenesis.Although NRF-12/2 blastocysts were morphologically normal,a striking decrease was observed in their mtDNA content. Thisdepletion of mtDNA occurred in the absence of a generalizedincrease in nuclear DNA fragmentation that may occur as aresult of increased apoptosis. The results are consistent with aspecific role for NRF-1 in maintaining mtDNA during earlyembryogenesis. The fact that NRF-12/2 embryos can surviveto the blastocyst stage with severely diminished mtDNA levelsis consistent with previous findings concerning the role of mi-tochondrial function during early embryogenesis. It has beendemonstrated that oxygen consumption starts to rise at theeight-cell stage and increased by 3.5-fold in the blastocysts(32), coinciding with changes in the mitochondrial ultrastruc-ture (35). Although drastic increases in mitochondrial rRNAand mRNA expression have been observed from two-cell em-bryos to blastocysts (37), mitochondrial genetic activities donot appear to be required for blastocyst development (35).Blastocysts that had been treated with inhibitors of mitochon-drial RNA and protein synthesis underwent normal develop-ment when transplanted to the uteri of foster mothers. Thus,although mitochondrial function may be necessary for embry-onic development to the blastocyst stage, normal expression ofthe mitochondrial genome may not be absolutely required. Inthe case of NRF-12/2 blastocysts, a low level of expressionfrom mtDNA combined with transcripts and proteins carriedfrom the oocyte may be sufficient to achieve this stage ofdevelopment.

mtDNA is massively amplified during mammalian oocytegrowth before the first meiotic devision (31), and no mtDNAreplication occurs from the egg through the blastocyst stage(36, 37). The reduced amount of mtDNA in homozygous blas-tocysts could thus be due to the effect of NRF-1 disruption onthe mtDNA amplification during oocyte growth, the mainte-nance of mtDNA during early embryogenesis, or a combina-tion of both. The positive b-galactosidase staining in the ovaryof heterozygous mice is consistent with the expression ofNRF-1 in growing oocytes, therefore suggesting a possible rolefor NRF-1 in mtDNA amplification. However, unfertilizedeggs from wild-type and heterozygous females had the sameamount of mtDNA, arguing against an abnormal mtDNA am-plification in heterozygous oocytes. Nonetheless, in addition totranscriptional regulation of the target genes in early embryos,NRF-1 may play a role in the normal maintenance of mtDNA

FIG. 6. Detection of chromosomal DNA fragmentation in blasto-cysts by TUNEL staining. TUNEL staining was performed on blasto-cysts as described in Materials and Methods. Blastocysts with ex-panded blastocoel cavities were selected to ensure that they hadentered the stage when limited apoptosis would normally occur.Bright-field microscopy (A, C, and E) and the corresponding fluores-cent images of TUNEL-stained blastocysts (B, D, and F) are shown. (Aand B) Wild-type blastocyst treated with RQ1 DNase as a positivecontrol. (C and D) Wild-type blastocyst showing very few stained cells.(E and F) NRF-12/2 blastocyst showing essentially the same stainingintensity as the wild-type embryos.



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in early embryogenesis by regulating the expression of genes ingrowing oocytes, whose products are essential when carriedinto the embryos.

Several NRF-1 target genes have been implicated in mito-chondrial maintenance. Among them, TFAM is required formtDNA transcription and replication (12, 34), and its expres-sion correlates with mtDNA levels in patients (28, 38) and inan in vivo animal model (29). NRF-1 binding was demon-strated to be important for human TFAM promoter activity invitro (49). Thus, it is plausible that the disruption of NRF-1may cause a reduction in mtDNA through reduced expressionof TFAM. However, TFAM-null mice die after embryonic day8.5, suggesting that the earlier death of NRF-1 homozygousembryos may not be due to the downregulation of TFAM. Onepossibility is that the phenotype is only in part contributed byTFAM, while involving other NRF-1-regulated mitochondrialgenes. Such candidates include MRP RNA, the RNA compo-nent of the endonuclease necessary for generating primerRNAs for mitochondrial transcription (6), and mtSSB, thesingle-stranded DNA-binding protein that binds to mtDNAD-loops and enhances the rate of the DNA polymerase reac-tion (17). These and other unidentified NRF-1 target genes,together with TFAM, may act in concert in mitochondrialmaintenance.

NRF-1 disruption and embryonic lethality. Could the re-duced amount of mtDNA in homozygous blastocysts be thecause of embryonic death? The mouse embryo does not growin size until the blastocyst stage and begins to grow rapidlyduring implantation (19). mtDNA synthesis is activated ataround this time (30). Therefore, although reduced mtDNAcontent could provide sufficient mitochondrial expression atthe blastocyst stage, the rapid growth of the embryo starting atimplantation may require more efficient energy production.Hence, the reduced number of mtDNA molecules may becomeinadequate, leading to cell death because of low levels of oxi-dative phosphorylation.

In light of the broader role for NRF-1 in transcriptionalregulation, it is unlikely that this simple model can adequatelyexplain the embryonic lethal phenotype. Since NRF-1 wasidentified, several genes whose functions are not directly re-lated to mitochondria have been found to be regulated byNRF-1 at the transcriptional level in vitro. Some of these geneproducts are involved in fundamental cellular functions, in-cluding amino acid metabolism (tyrosine aminotransferase),translation initiation (eIF-2a), chromatin structure (histoneh5), and purine synthesis (GPAT and AIRC) (reviewed inreference 39). It is possible that the disruption of NRF-1 func-tion causes their abnormal expression, and one or more ofthese proteins are essential for embryonic development. Con-ceivably, other unidentified NRF-1-dependent factors could berequired for embryogenesis. Computer searches revealed po-tential NRF-1 binding sites in a variety of mammalian genes ofdiverse functions (48). Null mutations of some of these genesin the mouse have demonstrated that they are essential forembryonic development. Examples include the mouse Dad1gene (defender against apoptotic cell death) (22), the mousecdh5 gene (vascular endothelial cadherin) (5), and the mouseLrp gene (low-density lipoprotein receptor-related protein)(20). Thus, the disruption of NRF-1 may lead to dysfunction ofa number of cellular pathways in addition to mitochondrial

deficiency via abnormal gene regulation. These effects may actin combination to cause embryonic lethality.

ACKNOWLEDGMENTS

We acknowledge the assistance of the Targeted Mutagenesis Facilityin the Children’s Memorial Institute for Education and Research atNorthwestern University. We are grateful to Jan Reddy for his exper-tise and advice. We thank Kristel Vercauteren for excellent technicalassistance and Ulf Andersson for critical reading of the manuscript.

This work was supported by United States Public Health ServiceGrant GM32525-18.

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